Project Summary/Abstract Cardiovascular disease is the major cause of death in Americans over the age of 65. Mechanisms of cardiovascular aging are not well defined nor are effective treatments available. This project proposes a novel hypothesis: age-related loss in the expression by cardiac myocytes of caveolae and the protein caveolin-3 with the resultant stimulation of pro-fibrotic activity of cardiac fibroblasts, thereby enhancing production of extracellular matrix, cardiac fibrosis, diastolic dysfunction, and heart failure, especially heart failure with preserved ejection fraction (HFpEF). HFpEF is a clinical problem for which no effective therapies exist. This hypothesis derives from our preliminary data which show that cardiac myocytes of aged mice have a loss in expression of caveolae and their key resident protein, caveolin-3 and that cardiac fibroblasts isolated from aged animals have increased pro- fibrotic activity, which we detect in fibroblasts cultured ex vivo. Our proposed studies will test if caveolin-3 in cardiac myocytes has cardioprotective properties in aging by reducing the profibrotic state of cardiac fibroblasts, thus blunting the development and progression of aging-related cardiac fibrosis. In addition, we will determine if restoration of the loss in caveolin-3 with advanced age can reduce age-related cardiac fibrosis. Our two specific aims will ask: 1) Does caveolin-3 expression in cardiac myocytes regulate the pro-fibrotic state of cardiac fibroblasts and cardiac fibrosis in aging? and 2) Does restoration of caveolin-3 in cardiac myocytes reduce age- related cardiac fibrosis? In Aim 1, we will assess cardiac function and the fibrotic activity of cardiac fibroblasts isolated from young (2-3 month) and aged (18-22 month) wild-type, caveolin-3- knockout, and cardiac myocyte- targeted caveolin-3 over-expressing mice. We will also conduct RNA-seq studies of cardiac myocytes and cardiac fibroblasts and will use conditioned media from cardiac myocytes to assess exosomes and soluble proteins released into the media by young and old hearts. In addition to studies of physiological aging, we will subject mice to transaortic constriction, which induces cardiac fibrosis, so as to increase the dynamic range of fibrotic changes. In Aim 2, we will test the impact of AAV constructs engineered to increase caveolin-3 expression in cardiac myocytes on cardiac function, cardiac myocytes and cardiac fibroblasts using the approaches from Aim 1. Based on our preliminary data and past efforts, the proposed studies should be highly feasible even though they are of the high risk/high reward type that is sought in R21 applications. We believe that the results will advance understanding of aging-related cardiac fibrosis and dysfunction and may identify a therapeutic approach?increasing caveolin-3 expression in the heart?for a major contributor to morbidity and mortality in the aged population.